Temperature and pH sensitive copolymers

ABSTRACT

The invention is directed to a copolymer comprising at least three types of monomeric units, said three types of monomeric units comprising:
         a temperature-sensitive unit,   a hydrophilic unit, and   a hydrophobic unit comprising at least one pH-sensitive moiety;
 
wherein said hydrophobic monomeric unit is derived from a copolymerizable unsaturated fatty acid.

This invention relates to novel copolymers, in particular, temperature-and pH-sensitive amphiphilic copolymers. The invention also relates tocompositions comprising novel copolymers which are useful for drugdelivery, as well as to methods of providing a selected therapeuticagent to an animal or human.

BACKGROUND OF THE INVENTION

The development of sophisticated pharmaceutical products and drugdelivery methods that can provide precise targeting, timing and dosingof therapeutic drugs has been necessitated in part by complexrequirements in the treatment of organ-specific disorders due todiseases such as cancer, HIV/AIDS, cystic fibrosis, etc. Some of thecontributing factors to the complex requirements of treatment includethe toxicity of drugs used in the treatment of such diseases, limitedtherapeutic activity of the drugs, as well as the inaccessibility andheterogeneity of the diseased organ.

Progress has been made in the delivery of drugs, particularly in thedevelopment of drug carriers showing low toxicity and which are capableof providing improved targeting of the diseased cells. Several types ofdrug carriers that provide improved drug delivery have beeninvestigated, including liposomes, drug-polymer conjugates andnanoparticles.

Polymeric core-shell nanoparticles have emerged recently as promisingcolloidal carriers for targeting poorly water-soluble and amphiphilicdrugs as well as genes to tumour sites [Kataoka et al.—Advanced DrugDelivery Rev. 47 (2001) 113-131; V. P. Torchilin—J. Control. Rel. 73(2001) 137-172; Allen et al.—Cool. Surf. B: Biointerfaces 16 (1999)3-27]. Polymeric core-shell nanoparticles are small in size, generallyless than 200 nm, and can solubilize hydrophobic drugs, genes orproteins in their inner cores through hydrophobic interaction,electrostatic interaction and hydrogen bonding etc., while exposingtheir hydrophilic shells to the external environment. This effectivelyprotects the enclosed bioactive compounds against degradation andenables them to exhibit prolonged activity in the systemic circulationby avoiding being scavenged by reticuloendothelial systems (RES). Withpolymeric core-shell nanoparticles, targeting can be achieved, bothpassively and actively, —through an enhanced permeation and retentioneffect (EPR effect) [Matsumura et al.—Cancer Research 46 (1986)6387-6392] and the incorporation of recognition signals onto the surfaceof the micelles [Kabanov et al.—FEBS Lett. 258 (1989) 343-345] orintroducing a polymer sensitive to variations in physiologicalenvironment such as temperature or pH.

Polymeric nanoparticles which have shells constructed fromtemperature-sensitive poly(N-isopropylacrylamide) (PNIPAAm) haverecently attracted considerable attention because of the polymer'sthermal responsiveness. PNIPAAm exhibits a lower critical solutiontemperature (LCST) of around 32° C. in aqueous solution, below which thepolymer is water-soluble and above which the polymer is water-insoluble[Taylor et al.—J. Polym. Sci.: Polym. Chem. Ed. 13 (1975) 2551-2570].The temperature-sensitivity of the polymer advantageously provides ameans to target drug carriers thermally.

Okano et al. reported the synthesis of adriamycin-incorporated micellarstructures derived from PNIPAAm-b-poly(butylmethacrylate) andPNIPAAm-b-poly(D,L-lactide) block copolymers [Chung et al.—J. Control.Rel. 62 (1999) 115-127; Kohori et al—Colloids and Surfaces B:Biointerfaces 16 (1999) 195-205]. The core-shell nanoparticles were wellformed below LCST, but deformed at temperatures higher than LCST. Therelease of the drug was regulated through a combination of local heatingand cooling cycles. However, it was found that temperature regulationalone was not efficient in targeting deep tissues or tumours.

One alternative to temperature sensitive drug carriers are pH-sensitivedrug carriers. It is known, for example, that the extracellular pH ofmost solid tumours range from 5.7 to 7.8 [Vaupel et al.—Cancer Research41 (1981) 2008-2013], while the pH of the tumour interstitial fluidrarely declines below pH 6.5. It is a challenge to provide a drugcarrier with such a narrow pH window [Drummond et al.—Progress in LipidResearch 39 (2000) 409-460].

Chen and Hoffman reported the synthesis of a copolymer of NIPAAm andacrylic acid and its pH-dependent LCST, and proposed its possibleapplication in drug targeting [Nature 373 (1995) 49-52]. More recently,core-shell nanoparticles made from poly(L-histidine)-b-poly(ethyleneglycol) (PEG) were reported to be pH-sensitive, which released theenclosed drug, doxorubicin (DOX), at pH from 7.4 to 6.8 [Lee et al.—J.Control. Rel. 90 (2003) 363-374; J. Control. Rel. 91 (2003) 103-113].The acidic environment triggered the destabilization of the core-shellnanoparticles and thus release the enclosed drug molecules at tumourtissues.

WO 01/87227 A2 discloses the use of a colloidal composition consistingof polymeric micelles having a hydrophobic core and a hydrophilic shell.The pH- and temperature-sensitive micelles are derived from a copolymerof NIPAAm, methacrylic acid and octadecyl acrylate. Thetemperature-sensitive and pH-sensitive moieties are located on the shellof micelles.

Despite the developments that have taken place, limitations in thecurrent drug carriers still exist for which continuing efforts areneeded to improve their performance.

Accordingly, it is an object of the present invention to providepolymeric compounds which can be used as drug carriers that haveimproved pH and temperature sensitivity, and thus provide improved drugdelivery performance.

SUMMARY OF THE INVENTION

The present invention provides pH and temperature sensitive copolymerswhich can be used as materials for drug delivery. In one aspect, theinvention is directed to a copolymer comprising at least three types ofmonomeric units, said three types of monomeric units comprising:

a temperature-sensitive unit,

a hydrophilic unit, and

a hydrophobic unit comprising at least one pH-sensitive moiety;

wherein said hydrophobic monomeric unit is derived from acopolymerisable unsaturated fatty acid.

In another aspect, the invention is directed to a temperature and pHsensitive composition comprising:

-   -   a therapeutic agent, and    -   a copolymer comprising at least three types of monomeric units,        said three types of monomeric units comprising:        -   a temperature-sensitive unit,        -   a hydrophilic unit, and        -   a hydrophobic monomeric unit comprising at least one            pH-sensitive moiety;    -   wherein said hydrophobic monomeric unit is derived from a        copolymerisable unsaturated fatty acid.

In yet another aspect, the invention provides a method of providing aselected therapeutic agent to an animal or human, comprisingadministering to said animal or human a temperature and pH-sensitivecomposition comprising:

-   -   a therapeutic agent, and    -   a copolymer comprising at least three types of monomeric units,        said three types of monomeric units comprising:        -   a temperature-sensitive unit,        -   a hydrophilic unit, and        -   a hydrophobic unit comprising at least one pH-sensitive            moiety;    -   wherein said hydrophobic monomeric unit is derived from a        copolymerisable unsaturated fatty acid;    -   wherein said copolymer is arranged into at least one        nanoparticle comprising a hydrophobic core and a hydrophilic        shell; and    -   wherein said therapeutic agent is contained within said        hydrophobic core.

These aspects of the invention will be more fully understood in view ofthe following description, drawings and non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Scheme 1, which illustrates one possible scheme forcarrying out the synthesis of a copolymer of the invention, using themonomers N-isopropylacrylamide, N,N′-dimethylacrylamide and10-undecenoic acid, and a chain transfer agent, aminoethanethiol, forillustrative purposes. Table 1 shows the various feed molar ratios usedto form copolymers of the invention in exemplary experiments, and thephysical characteristics of the resulting copolymers, namely, molecularweight, glass transition temperature, actual molar ratio of hydrophilicunits to temperature-sensitive units, acidity, and the thermaldegradation temperature. Aminoethanethiol was used as a chain transferagent to introduce a terminating moiety into these copolymers.

FIG. 2 depicts a typical ¹H NMR spectrum of a polymer obtained frommonomers N-isopropylacrylamide, N,N′-dimethylacrylamide and10-undecenoic acid synthesised from a feed molar ratio of 3.5:1.5:0.5(Polymer III) in CDCl₃.

FIG. 3 depicts a typical FT-IR spectrum of Polymer III.

FIG. 4 depicts a plot of optical transmittance of the polymer obtainedfrom monomers N-isopropylacrylamide, N,N′-dimethylacrylamide and10-undecenoic acid synthesised from a feed molar ratio of 4.0:1.00:0.5(Polymer I) as a function of temperature at varying pH at 500 nm.

FIG. 5 depicts a plot of transmittance of the polymer obtained frommonomers N-isopropylacrylamide, N,N′-dimethylacrylamide and10-undecenoic acid at a molar ratio 3.75:1.25:0.5 (Polymer II) as afunction of temperature at varying pH at 500 nm.

FIG. 6 depicts a plot of transmittance of Polymer III as a function oftemperature at varying pH at 500 nm.

FIG. 7 depicts a plot of transmittance of Polymer II as a function oftemperature in PBS (pH 7.4) with 10 (w/v) % BSA at 500 nm.

FIG. 8 depicts a plot of I₃/I₁ as a function of polymer concentration(Polymer II).

FIG. 9 depicts a typical presentation of the size distribution ofDOX-loaded nanoparticles.

FIG. 10 depicts a TEM picture of drug-loaded nanoparticles.

FIG. 11 shows the cytotoxicity of polymer II against L929 cells.

FIG. 12 shows the release profiles of DOX from Polymer II nanoparticlesat varying pH at 37° C.

FIG. 13 shows Scheme 2, which illustrates the activation of folic acidwith N-hydrosuccinimide (NHS).

FIG. 14 shows Scheme 3, which illustrates the conjugation of folic acidto Polymer II.

FIG. 15 shows Scheme 4, which illustrates the conjugation of cholesterolto polymer II via NHS activation.

FIG. 16 shows the NMR spectrum of folic acid-conjugated Polymer II.

FIG. 17 shows the NMR spectrum of cholesterol-grafter Polymer II.

FIG. 18 shows the optical transmittance of poly-folic acid and PolymerII as a function of temperature at various pH at 500 nm.

FIG. 19 shows the optical transmittance of poly-cholesterol as afunction of temperature at a pH of 7.4 at 500 nm.

FIG. 20 shows the optical transmittance of poly-cholesterol-folic acidas a function of temperature at different pH at 500 nm.

FIG. 21 shows bar charts depicting the cytotoxicity ofpoly-cholesterol-folic acid against L929 cells.

FIG. 22 shows the DSC analyses of the copolymer, paclitaxol andpaclitaxol-loaded core-shell nanoparticles.

FIG. 23 shows the structural formula of doxorubicin hydrochloride.

FIG. 24 shows the synthesis of doxorubicin conjugated Polymer II.

FIG. 25 shows the gel permeation chromatogram of doxorubicin conjugatedPolymer II.

FIG. 26 shows the differential scanning calorigram of doxorubicinconjugated Polymer II.

FIG. 27 shows the temperature sensitive reversible particle size of adrug conjugated micelle in PBS at a pH of 7.4.

FIG. 28 shows Scheme 5, which illustrates the synthesis of thetemperature block consisting of NIPAAm and DMAAm, and the synthesis ofpoly(10-undecenoic acid).

FIG. 29 shows Scheme 6, which illustrates the synthesis of a blockcopolymer.

FIG. 30 shows the gel permeation chromatogram of temperature sensitiveblock and of the block copolymer.

FIG. 31 shows the LCST measurements made of the block copolymers.

DETAILED DESCRIPTION

The present invention is based on the finding that core-shellnanoparticles obtained from copolymers which incorporate fatty acids ashydrophobic, pH-sensitive functionalities possess excellentcharacteristics as drug carriers. The copolymers of the invention canself-assemble into a core-shell structure comprising a hydrophobic core,in which hydrophobic moieties such as the fatty acid are arranged,surrounded by a hydrophilic shell comprising hydrophilic moieties of thecopolymer. Drugs can be encapsulated by either physical entrapment orchemical conjugation within the core. These nanoparticles are able toalter their physical configuration in response to a narrow window ofchange in environmental pH, resulting in the liberation of the drugencapsulated within the hydrophobic core of the nanoparticles.

One advantage of the copolymers of the invention is that the lowercritical solution temperature (LCST) of core-shell nanoparticles formedfrom these copolymers can be made to be dependent on the environmentalpH, meaning that structural deformation of the core-shell nanoparticlescan be triggered by environmental pH changes. This property can beharnessed for use in targeting organs or tumour tissues where theenvironment is characteristically acidic. Under normal physiological pH,the core-shell nanoparticles have an LCST that is above normal bodytemperature (about 37° C.). However, in slightly acidic environments,the LCST of the nanoparticles is lower than the normal body temperature.This means that the core-shell nanoparticles are stable in thephysiological environment but destabilise or aggregate in acidicenvironments.

Without wishing to be bound by theory, it is believed that the use offatty acids as the hydrophobic, pH-sensitive portion of the copolymerhelps to provide greater pH sensitivity in the copolymer of theinvention. The pH sensitive functionalities are bound to hydrophobicsegments of the fatty acid, meaning that they are assembled into thecore of core-shell nanoparticles. It is also believed that core-shellnanoparticles made from these polymers are loosely packed, so that thepH sensitive functionalities remain accessible to the externalenvironment despite being located at the core of the nanoparticles. Whenthe pH of the external environment is changed, the pH sensitivefunctionalities can also be changed, e.g. ionised or deionised. Thisleads to changes to the hydrophobicity of the fatty acid, and thusalters the LCST of the nanoparticles, resulting in the release of drugmolecules. Furthermore, as fatty acids are natural compounds, they arealso believed to be highly biocompatible and should thus exhibit verylow levels of toxicity within the human body.

The copolymer of the invention comprises at least three types ofmonomeric units. It is herewith mentioned for clarity that the term‘monomeric unit’ refers to a monomer that has been polymerised into apolymer. It is distinguished from the term ‘monomer’, which denotes adistinct molecular entity which can be polymerised into a polymer.

One type of monomeric unit required in the copolymer of the invention isa temperature sensitive/responsive unit. In the present invention,temperature sensitive monomeric units are used to impart temperaturesensitivity to the copolymers, resulting in the formation oftemperature-sensitive copolymers. Temperature sensitive copolymerstypically exhibit a distinct LCST or UCST (upper critical solutiontemperature), also known as phase transition temperature. Copolymershaving a distinct LCST (hereinafter known as ‘LCST systems’) areinsoluble in water above the LCST, while those having a distinct UCSTare insoluble below the UCST. This characteristic is evidenced byconformational changes in the copolymer either when temperature changeoccurs across the critical solution temperature, or when the criticalsolution temperature of the copolymer shifts across a static environmenttemperature in response to pH changes, for example. In general, mostdrug delivery applications utilise LCST systems. The abrupt shrinkingand the resulting insolubility of LCST systems when the environmentaltemperature is above the LCST allows the copolymer of the invention toleave the aqueous phase and assume a hydrophobic phase, therebyfacilitating interaction with cell membranes. Furthermore, temperaturesensitive monomeric units can be co-polymerized with hydrophilicco-monomers such as acrylamide (AAm), or other types of modifyingco-monomers to achieve a higher or lower LCST. Such copolymers can beapplied as a functional drug delivery material for controlling drugrelease rate.

Temperature sensitive monomeric units suitable for use in the inventionmay possess one or more polar functionalities, such as a primary,secondary or tertiary amino group, an amide group, a carboxyl group, acarbonyl group, or a hydroxyl group, all of which are polar by nature.Generally, due to the presence of polar functionalities, temperaturesensitive monomeric units are consequently also hydrophilic in nature.Examples of temperature sensitive monomeric units that may be used inthe invention include monomeric units derived from monomers such assubstituted acrylamides, acrylates, pyrrolidone, piperidine, andcellulose. Specific examples of suitable temperature-sensitive monomericunits include, but are not limited to, those derived fromN-isopropylacrylamide (NIPAAm), N-hydroxypropyl acrylate,N-acryloylpyrrolidone (APy), N-acryloylpiperidine, N-acroylpiperadine,hydroxy-methylcellulose, N-t-butylacrylamide,N-piperidyl-methacrylamide, for example. A presently preferred monomerthat for used as the temperature sensitive monomeric unit of theinvention is NIPAAm. Polymers incorporating monomeric units of NIPAAmare highly temperature sensitive, and display negative temperaturesensitivity (i.e. LCST system), meaning that they become water solubleat temperatures falling below its LCST.

Another type of monomeric unit required in the present copolymer is ahydrophilic unit. In general, the hydrophilic monomeric unit provides ameans to modify/shift the LCST of the copolymer of the invention. Whenthe hydrophilic unit is relatively more hydrophilic than thetemperature-sensitive hydrophilic unit, the LCST of the copolymer may beincreased; conversely, if a relatively less hydrophilic unit or ahydrophobic unit is present, the LCST of the copolymer may be lowered.Hydrophilic monomeric units suitable in the invention include anysuitable copolymerisable monomer, which may have one or more polarfunctionalities, such as a primary, secondary or tertiary amino group,an amide group, a sulfhydryl group, a carboxyl group, a carbonyl group,or a hydroxyl group. As opposed to the temperature sensitive monomericunit in which polar functionalities may also be present and may thusalso be hydrophilic in nature, hydrophilic monomeric units required inthe invention do not have to be temperature-sensitive.

In certain embodiments, the hydrophilic monomeric unit is relativelymore hydrophilic than the temperature sensitive hydrophilic unit. Thisserves to increase the LCST of the resulting copolymer. Hydrophilicmonomeric units present in these embodiments may be derived from, butare not limited to, the following monomers: acrylic acid, acrylamide,acrylate, pyrrolidone, ethylene glycol and derivatives thereof. Inspecific embodiments, the hydrophilic monomeric unit can be derived fromacrylamide and N-substituted acrylamide derivative monomers, including,but not limited to, acrylamide (Mm), N,N′-dimethylacrylamide (DMAAm),and N-(hydroxymethyl)acrylamide. In a preferred embodiment in whichDMAAm is present in the copolymer of the invention, thermosensitivitywas enhanced, thereby enabling ‘on-off’ drug release in response tosmaller temperature changes in the body temperature. Additionally, withNIPAAm and DMAAm present in the copolymer of the invention, the LCST ofthe copolymer of the invention was raised (to a temperature slightlyabove 37° C.). It is generally desirable to utilise polymers in whichthe LCST of the copolymer is slightly above body temperature underphysiological conditions. It is believed that no upper limit for theLCST is required, as long as the change in environmental pH from normalphysiological pH (typically 7.4) to 7.2 or less is able to shift theLCST from a value higher than normal body temperature to a value lowerthan normal body temperature.

A third type of monomeric unit required in the copolymer of theinvention is a hydrophobic unit derived from a copolymerisableunsaturated fatty acid and which comprises at least one pH-sensitivemoiety. Any suitable unsaturated fatty acid which possesses at least onepH-sensitive functionality can be used in the invention. Suitableunsaturated fatty acids include all natural and artificiallymodified/synthesised fatty acids, and all monounsaturated andpolyunsaturated fatty acids having 1, 2, 3, 4, 5, 6, 7, 8 or morecarbon-carbon double and/or triple bonds, as well as all cis- andtrans-isomers thereof. The unsaturated portion of the fatty acid, i.e.the carbon-carbon double bond, may be present at any location in maincarbon chain of the fatty acid.

The hydrocarbon chain of the unsaturated fatty acid constitutes the mainhydrophobic portion of the copolymer. The hydrophobic portion isresponsible for imparting hydrophobic nature to the copolymer of theinvention. Hydrophobic portions are needed to form a core-shellstructure and enable the copolymer to interact with other hydrophobicmaterials, such as anticancer drug molecules. The hydrocarbon chain ofsuitable fatty acids may be straight, unbranched alkyl chains typicallyfound in natural fatty acids (including branched alkyl chains). It mayalso be cyclic or branched alkyl chains, optionally substituted withfunctional groups such as carboxylic acid, amine or hydroxyl groups, forexample. No restriction is placed on the position of the functionalgroups. The carboxylic acid group provides, amongst other things,pH-sensitivity to the copolymer as well as the ability for conjugationwith suitable ligands. Any other pH-sensitive functionality present inthe fatty acid may also serve these functions.

In one embodiment, the copolymer of the invention has the followingstructural formula (I):

A, B and C, as defined in the above formula, depict the respectiverandomly co-polymerised monomeric units, namely, temperature-sensitiveunit, hydrophilic unit and hydrophobic unit, or polymer blocks thereof.X denotes a carboxylic acid functional group which is directly bonded tothe hydrophobic segment of the fatty acid.

In another embodiment, the unsaturated fatty acid comprises between(inclusive of 5 to 50 or more main chain carbon atoms. In thisembodiment, the fatty acid may comprise a single carbon-carbon doublebond, meaning that it is a mono-unsaturated fatty acid. Specificmonomers from which the hydrophobic monomeric unit can be derivedinclude, for example, pentenoic, hexenoic, heptenoic, octenoic,nonenoic, decenoic, undecenoic, and dodecenoic acids. No specificrestriction is placed on the position of the carbon-carbon double bondin the fatty acid.

Presently preferred mono-unsaturated fatty acids include, but is notlimited to, fatty acids selected from the group consisting of(Z)-9-Tetradecenoic acid, (E)-9-Hexadecenoic acid, (Z)-9-Hexadecenoicacid, (E)-9-Octadecenoic acid, (Z)-9-Octadecenoic acid,(Z)-11-Octadecenoic acid, (Z)-11-Eicosenoic acid, (Z)-13-Docosenoic Acidand (Z)-15-Tetracosaenoic Acid.

In one embodiment, the monounsaturated fatty acid is an omega-1 fattyacid, meaning that the double bond is present between the first andsecond carbon atom at the end of the fatty acid that is opposite to thelocation of the carboxylic acid functional group. An advantage in usingomega-1 fatty acids is that it enables the fatty acid to be readilycopolymerised with the required temperatures-sensitive and hydrophilicmonomeric units, as the carbon-carbon double bond is not stericallyhindered by bulky alkyl chains. Presently preferred omega-1 fatty acidsare selected from the group consisting of 4-pentenoic acid, 7-octenoicacid, 10-undecenoic acid, 15-hexadecenoic acid, and 19-eicosenoic acid.

In another embodiment, the fatty acid comprises at least 2 carbon-carbondouble bonds, meaning that the fatty acid is polyunsaturated. Suitablepolyunsaturated fatty acids include omega-3, omega-6 and omega-9 fattyacids as well as other types of fatty acids. Specific examples ofpolyunsaturated fatty acids which can be used in the invention include(E,E)-9,12-Octadecadienoic acid, (Z,Z)-9,12-Octadecadienoic acid,(E,E)-9,11-Octadecadienoic acid, (Z,Z,Z)-9,12,15-Octadecatrienoic acid,(Z,Z,Z)-6,9,12-Octadecatrienoic acid,(Z,Z,Z,Z)-6,9,12,15-Octadecatetraenoic acid, (Z,Z,)-11,14-Ecosadienoicacid, (Z,Z,Z)-5,8,11-Eicosatrienoic acid,(Z,Z,Z)-11,14,17-Eicosatrienoic acid, (Z,Z,Z)-8,11,14-Eicosatrienoicacid, (Z,Z,Z,Z)-8,11,14,17-Eicosatetraenoic acid,(Z,Z,Z,Z)-5,8,11,14-Eicosatetraenoic acid,(Z,Z,Z,Z,Z)-5,8,11,14,17-Eicosapentaenoic acid,(Z,Z)-13,16-Docosadienoic acid, (Z,Z,Z)-13,16,19-Docosatrienoic acid,(Z,Z,Z,Z)-7,10-13-16-Ocosatetraenoic acid,(Z,Z,Z,Z,Z)-4,7,10,13,16-Docosapentaenoic acid,(Z,Z,Z,Z,Z)-7,10,13,16,19-Docosapentaenoic acid,(Z,Z,Z,Z,Z,Z)-4,7,10,13,16,19-Docosahexaenoic acid, and(Z,Z,Z,Z,Z,Z)-6,9,12,15,18,21-Tetracosahexaenoic acid.

Copolymers of the present invention can comprise only theabove-mentioned 3 types of monomeric units, or it may additionallyinclude other types of monomeric units. For example, it is also possibleto utilise two or more temperature-sensitive monomeric units, such asNIPAAm and N-t-butylacrylamide, or NIPAAm andN-piperidyl-methacrylamide. It is likewise possible to utilise two ormore hydrophilic monomeric units, such as DM m and AAm, or DMAAm andAPy. Other types of monomeric units can also be incorporated into thecopolymer backbone to adjust the physicochemical properties of thecopolymer, or introduce functional groups or spacers for furtherconjugation with ligands, these monomeric units includingN-(hydroxymethyl)acrylamide or heterobifunctional PEG, for example.

Copolymers of the invention can be random copolymers in which the threemain types of monomeric units, namely temperature-sensitive monomericunit, hydrophilic monomeric unit and hydrophobic unit, are randomlydistributed in the copolymer. It is also possible that the copolymer canbe synthesised as a block copolymer such as a diblock or triblock blockcopolymer, as well as a block-graft copolymer. In one embodiment, thetemperature-sensitive monomers and the hydrophilic monomers arecopolymerised to form one block of polymers and the hydrophobic monomeris copolymerised to form another block of polymers, thereby forming adiblock copolymer.

In one embodiment, the copolymer further comprises at least one terminalgroup. The terminal group comprises at least one moiety selected fromthe group consisting of a terminating moiety, a ligand, a drug molecule,a tag, a radioimmunoconjugate, a moiety for modifying thephysico-chemical characteristics of the copolymer, and a spacer(linker).

In a specific embodiment, the copolymer of the invention comprises aterminal group that consists of a terminating moiety having thestructure according to formula (II):

wherein Y is the terminating moiety.

In this embodiment, the terminal group is bonded to a terminal carbonatom in the carbon backbone (i.e. the carbon chain linking the monomericunits) of the copolymer. Typically, the terminating moiety can beintroduced into the copolymer by adding a chain transfer agentcontaining the desired terminal structure to a mixture of reactingmonomers, or it can be generated by living polymerisation methods. Chaintransfer agents are able to stop the growth of a growing polymer chainby providing a ‘quenching’ atom to the active radical at the end of thegrowing chain. It in turn is left as a radical which can attackunreacted monomers and thus initiate the growth of a new chain.Accordingly, chain transfer agents can be used in the present inventionto provide a suitable reactive functional group to a copolymer, as wellas to obtain low molecular weight polymers. Examples of chain transferagents include chloroform, carbon tetrachloride, aminoethanethiol,alkyl-mercaptans, octanethiol, decanethiol, n-dodecanethiol ort-dodecanethiol, mercapto-propionic acid, mercapto-succinic acid,thioglycolic acid, mercaptoethanol secondary alcohols thereof, alkylhalides, salts of phosphorus acids with an oxidation number less than 5,as well as other additives/chain limiters known to the skilled person.Other examples of chain transfer agents which can be used in theinvention include solvents, impurities, or suitable modifiers.

In a further embodiment, the terminating moiety comprises at least onefunctional group selected from the group consisting of hydroxyl,carboxyl, carbonyl and amino functional groups. Amino functional groupsare presently preferred, including primary or secondary amino groups.Amino groups can for example be present in an alkylthiol group that isbonded to the terminus of the copolymer, e.g. 2-amino-ethanethiol or2,2-diamino-ethanethiol. The presence of amino groups in the terminatingmoiety allows modifications on the polymer and the targeting group to bemade, for example, through the conjugation with ligands including, butnot limited to, small targeting molecules (e.g. folic acid, othervitamins and acetylcholine etc.), proteins (e.g. transferrin andmonoclonal/polyclonal antibodies etc.), peptides (e.g. TAT) andcarbohydrates (e.g. galactose and polysaccharides), which may berecognised by specific receptors at desired cells, tissues or organs. Inaddition, the amino groups can also be conjugated with drugs or tags(e.g. fluorescent probes for visualization or purification ofnanoparticles in a biological system of interest) orradioimmunoconjugates or chemical moieties to modify the polymerproperties, for example attaching hydrophobic segments to increasehydrophobicity of the polymer.

It will be appreciated by the skilled person that the terminating moietyY can be adapted to be a functional group that can react with functionalgroups present in ligands or tags or radioimmunoconjugates or drugs orother chemical moieties such as proteins. For example, where one or morecarboxylic acid functional groups are present in the selected ligand, aterminating moiety having an amino group can be used for facilitatingconjugation. Conversely, biological molecules that contain amino groupscan be attached to a terminating moiety having a carboxylic acidfunctional group.

In order to provide a suitable functionality for bio-recognition of atarget receptor, the copolymer may be conjugated to one or more ligandscapable of binding to functional groups present on the copolymer.Ligands which may be used include, but not limited to small targetingmolecules, proteins, peptides and carbon hydrates.

In one embodiment, the terminal group consists of a terminating moietyand a ligand. Copolymers carrying ligands can be used to efficientlytarget a desired tissue in the body or specific types or compartments ofcells. The targeting efficiency of the copolymer is enhanced by the pHsensitivity of the present copolymers.

Ligands which can be used in conjunction with copolymers of the presentinvention include, but are not limited to, small targeting molecules(e.g. folic acid, other vitamins and acetylcholine etc.), proteins (e.g.transferrin and monoclonal/polyclonal antibodies etc.), peptides (e.g.TAT) and carbon hydrates (e.g. galactose and polysaccharides). Thenumber of biologically active ligands that can be present in a singlecopolymer can range from 1, 2, 3, 4, 5 or more in number.

Illustrative examples of growth factors (proteins and peptides) whichare contemplated for use in the invention include Vascular EndothelialGrowth Factor (VEGF), Epidermal Growth Factor (EGF), Platelet-DerivedGrowth Factor (PDGF), Fibroblast Growth Factors (FGFs), TransformingGrowth Factors-b TGFs-b), Transforming Growth Factor-a (TGF-a),Erythropoietin (Epo), Insulin-Like Growth Factor-I (IGF-I), Insulin-LikeGrowth Factor-II (IGF-II), Interferon-g (INF-g), Colony StimulatingFactors (CSFs) are. Cytokines (proteins) that are contemplated for useinclude both lymphokines as well as monokines, and examples includeInterleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6),Interleukin-8 (IL-8). If cancer is the disease to be treated, theselected ligand should preferably be recognised by a specific receptoron the cancer cells. Specific types of cancer cell receptor ligands thatare contemplated for use with the copolymer of the invention includefolic acid, targretin, alitretinoin, E. coli toxin, C3 cleavagefragments (C3d, C3dg and iC3b), Epstein-Barr virus gp350/220 and CD23can be conjugated with the copolymers of the invention. Antibodies canalso be used as cancer cell ligands, including monoclonal and polyclonalimmunoglobulins obtained from mice, rabbits, chicken, goats and sheepand recombinant antibodies such as Fu fragments, scFu fragments, Fabfragments, or diabodies which are known to the skilled person. Cytokinesthat are contemplated for use include the TNF family of cytokines,including Tumour Necrosis Factor-a (TNF-a), Tumour Necrosis Factor-b(TNF-b), Fas Ligand (FasL) and TNF related apoptosis-inducing ligand(TRAIL). Other suitable ligands include transferrin, acetylcholine,biotin labels and folic acid. Prior modification of the ligand or of thecopolymers of the invention (e.g. by incorporating functional groupsthat can react with complementary functional groups on the ligand) canbe performed, if necessary.

In another embodiment, the terminal group is selected from the groupconsisting of a terminating moiety, a ligand, a tag, a drug molecule, aradioimmunoconjugate, or any other chemical moiety, e.g. a moiety formodifying the physico-chemical characteristics of the copolymer.

Copolymers incorporating a ligand, a drug, a tag, a radioimmunoconjugateor any other chemical moiety may have a general structure according toformula (III):

The terminating moiety Y can be bonded to a ligand, a tag, a drug, aradioimmunoconjugate, or a chemical moiety denoted by Q as shown in theabove formula.

The ligands, tags, drug, radioimmunoconjugates, or chemical moieties arenot limitedly bonded to the terminating moiety. In other embodiments,the ligands, tags, drugs, radioimmunoconjugates or chemical moieties maybe coupled or conjugated to functional groups located on the temperaturesensitive units or hydrophilic units. The terminal group mayalternatively be bonded to any one of the monomeric units, such as afunctional group in the hydrophilic unit or the temperature sensitiveunit, instead of the terminating moiety Y. In such embodiments, theterminating moiety may or may not be present. This embodiment is shownin the following formula (IV) and (V):

In this embodiment, P can be a ligand, a tag, a drug, aradioimmunoconjugate, or a chemical moiety.

In another embodiment, a hydrophobic molecule P′, such as a drugmolecule (e.g. doxorubicin) or a moiety for modifying the hydrophobicityof the copolymer, is conjugated to X. In so doing, the copolymer can bearranged into a core shell structure in which the hydrophobic moleculeis located in the core (formula (VI)):

In the embodiments shown in the above formulas (III), (IV), (V) and(VI), a spacer -s- may optionally be positioned between the ligand orthe tag or the radioimmunoconjugate or the drug or the chemical moleculeand the terminating moiety (formula IIIs):

or optionally between B and P (see formula IVs):

or optionally between A and P (see formula Vs):

or optionally be positioned between P′ and the carboxyl group (seeformula VIs):

It is also possible for P, P′ and Q, respectively, to be conjugated toboth the terminating moiety (Y) and the functional groups of thehydrophilic monomeric units B, or B and X, or X and Y, or X and A, or Yand A, or A and B, or any three of A, B, X and Y in a single copolymer,as exemplified in the following formula (VII):

Each spacer -s- in the above formula (VII) can be the same or different.

The spacer can have any suitable length or number of main chain atoms,as long as the ligand or the tag or the radioimmunoconjugate is freelyaccessible to cells or tissues or organs after the self-assembly of thecopolymers into the nanoparticles.

In preferred embodiments, one spacer is used, said spacer comprisingmore than 10 main chain atoms. An example of such a spacer can, forexample, be derived from polyoxyalkylene compounds such as poly(ethyleneglycol) and poly(propylene glycol). The ligand or the tag or theradioimmunoconjugate can be present at any position on the spacermolecule. In one embodiment, the ligand or the tag or theradioimmunoconjugate is bonded to functional groups positioned at aterminal main chain atom of the spacer molecule. In other embodiments,the ligand or the tag or the radioimmunoconjugate is bonded tofunctional groups located in any one of the side chains, of the spacermolecule, if present.

Copolymers of the present invention can be advantageously employed as amaterial for drug delivery, especially for the delivery of drugs whichare hydrophobic in nature. As the copolymers are amphiphilic in natureand as the water solubility of the copolymer can be manipulated bytemperature and/or pH changes, hydrophobic drugs can be convenientlypackaged in a core-shell structure, typically known as a core-shellnanoparticle, using the present copolymers. When the composition of theinvention is prepared in the aqueous phase below the LCST of thecopolymer, the copolymer and the hydrophobic drug will self-assembleinto a core-shell arrangement, whereby the hydrophobic drug ispositioned in the core, where it will interact with the hydrophobicsegments of fatty acid monomeric units. The temperature-sensitive andhydrophilic monomeric units will be positioned in the shell, interactingwith the solvent (water) molecules or other polar molecules and therebyrendering the hydrophobic drug soluble in the aqueous phase. In thisway, hydrophobic drugs can be rendered water soluble and can betransported in the blood stream. Since there are carboxylic acid groupspresent in the hydrophobic segments (i.e. fatty acid units), hydrophilicdrugs, proteins or peptides can also be encapsulated within the core ofthe nanoparticles, protecting the enclosed drugs, proteins and peptidesagainst degradation and enabling them to exhibit prolonged activity inthe systemic circulation by avoiding the scavenging of thereticuloendothelial systems (RES). When the drug is packaged in acore-shell nanoparticle, it forms a composition, which can be readilyadministered to a patient, either intravenously, orally,intramuscularly, topically, or through the ocular route or inhalation.In general, there is no restriction to the molecular weight of thecopolymer that is used to form the nanoparticle. However, it ispreferable to keep the molecular weight of the copolymer to less than40,000 in order for the polymer to be excreted through the kidney.Several parameters may influence the size of nanoparticles formed usingthe present copolymer, including polymer concentration, drug loadinglevel and fabrication conditions of the nanoparticles. The size ofnanoparticles synthesised from copolymers disclosed herein may typicallybe less than 200 nm, particularly for enhanced permeability andretention (EPR) effect and long circulation in plasma.

Each of the at least three monomeric units can be present in copolymersof the invention in any suitable ratio. In general, the ratio ofmonomeric units can be varied to achieve desired LCST and pHcharacteristics. The ratio also depends on other factors such as thetype and number of functional groups present in each monomeric unit andthe pH- and temperature-sensitivity of the copolymer. The molar ratio ofmonomeric units present in the copolymer largely depends on the feedmolar ratio of each monomeric unit used to prepare the copolymer.

In one embodiment, the molar quantity of temperature-sensitive monomericunit present in the copolymer of the invention is larger than that ofthe hydrophilic monomeric unit to avoid dilution effect of thehydrophilic unit on the temperature-sensitivity of the final copolymer;the molar quantity of hydrophilic monomeric unit is in turn is largerthan the amount of hydrophobic monomeric unit in the copolymer. Incertain embodiments, the feed molar quantity of temperature-sensitivemonomers used to prepare copolymers of the invention is between about 2to 6 times more than the feed molar quantity of hydrophilic monomers,and between about 4 to 8 times more than the feed molar quantity ofhydrophobic monomers. Examples of feed molar ratios that can be used toform useful copolymers are 1 to 4 molar ratio of temperature sensitivemonomer; 0.5 to 1.5 molar ratio of hydrophilic monomer; 0.01 to 0.75molar ratio of hydrophobic monomer. In a particularly suitableembodiment, the respective monomers that were used according to thisrange of feed molar ratios are N-isopropylacrylamide,N,N′-dimethylacrylamide and 10-undecenoic acid.

In one preferred embodiment, the LCST of the core-shell nanoparticles isless than 37° C. at a pH of less than 7, or preferably less than 7.2. Inanother embodiment, the lower critical solution temperature of thecore-shell nanoparticles is higher than 37° C. under the normalphysiological conditions (pH 7.4). It is noted that the LCST of thecopolymer can be controlled, either raised or lowered, by varying thepercentages of monomeric units or the nature of hydrophilic monomericunits used to form the copolymer. It is presently preferred to have acopolymer in which the LCST of the core-shell nanoparticles at aphysiological pH is above the normal body temperature, i.e. 37° C., andthe LCST is below the normal body temperature in an acidic environment.Reversible, pH-dependent LCST and phase transition characteristics aredisplayed by nanoparticles formed using copolymers of the presentinvention. For example, in one specific experimental set up, in whichcore-shell nanoparticles were self-assembled in aqueous solutions fromthe copolymer of N-isopropylacrylamide, N,N′-dimethylacrylamide and10-undecenoic acid in a mole ratio of 3.75:1.25:0.5, the LCST of thecore-shell nanoparticles formed at this specific composition was 38.5°C. in phosphate-buffered saline (PBS, pH 7.4), which decreasedsignificantly (35.5° C.) in a slightly acidic environment (e.g. lessthan pH 6.6).

Drugs which have been contemplated for use in the invention include, butare not limited to, anti-cancer drugs, anti-inflammatory drugs, drugsfor treating nervous system disorders, and immunosuppressants etc. Forexample, doxorubicin, anastrozole, exemestane, cyclophosphamide,epirubicin, toremifene, letrozole, trastuzumab, megestrol, nolvadex,paclitaxel, docetaxel, capecitabine, goserelin acetate, cyclosporin,cisplatin, indomethacin, betamethason and doxycycline.

For efficient drug delivery, it is advantageous for the drug carrier topromote lesion targeting and intracellular access. Targeting, andsubsequent internalization under certain conditions of the drug carriersmay be achieved by coupling the drug carrier with a normally endocytosedligand, taking advantage of the natural endocytosis pathway. Using thisstrategy, copolymers of the invention can be incorporated with a varietyof ligands, such as monoclonal antibodies, growth factors, or cytokines,can be used to facilitate the uptake of carriers into target cells.Small and non-antigenic ligands are presently contemplated for use inthe invention in order to avoid difficulties in diffusion throughbiological barriers, e.g. cell walls of the target cell, as well asimmunogenecity.

In one embodiment, folic acid is used as the ligand. Folic acid (MW=441Da) is a low molecular weight and non-antigenic ligand, good as atargeting signal of tumour cells. Folic acid is a vitamin whose receptoris frequently expressed on the surface of the human cancer cells.Additionally, it exhibits very high affinity for its cell surfacereceptor (Kd˜10-10 M) and it can move into cytoplasm. Folic acid hasbeen found to follow the caveolae-mediated endocytosis, rather than endup in lysosomes, where the contents are rapidly degraded. Thus, by usingfolic acid as a ligand in drug compositions using copolymers disclosedherein, drugs can be delivered to a desired intracellular locality, safefrom degradative enzymes. It is also known that endosomes reachable viathe caveolae pathway are also acidic. Due to their acidity, endosomescan alter the LCST of nanoparticles (from a temperature higher than bodytemperature to a temperature lower than body temperature), breaking downthe endosome membrane. Therefore, intracellular drug delivery into thecytoplasm can be achieved.

It is to be noted that the compositions to which invention is directedis not limited to core-shell structured nanoparticles in which thetherapeutic drug is loaded into the hydrophobic core of thenanoparticles via hydrophobic interaction with the hydrophobic moietiesin the copolymer. It is possible to covalently bond molecules of thetherapeutic drug to the copolymer by reacting the drug molecule tosuitable functional groups located on any part of the copolymer, such asby reacting the carboxyl groups on fatty acids with amino groups orhydroxyl groups on the drug molecule, for example. Compositions in whichthe drug molecule is conjugated with the copolymer may assume bothcore-shell structures or micellar structures or any other stablestructure suitable for facilitating drug delivery. The conjugation ofdrugs to the copolymer of the invention provides an alternative butsimilarly effective means of delivering drugs to a target organ/celllocation.

In another embodiment in which doxorubicin is used as the therapeuticdrug in a composition of the invention, doxorubicin was conjugated tothe copolymer through carbodiimide chemistry. The amine functional groupof doxorubicin was conjugated to carboxyl functional groups of acopolymer of the invention, as presented in FIGS. 23 and 24.

The following examples are offered in order to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

Example 1 Synthesis ofpoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-10-undecenoicacid) [P(NIPAAm-co-DMAAm-co-UA)] A) Experimental Section

i) Materials

Unless stated otherwise, all reagents and solvents were of commercialgrade, and were used as received. N-isopropylacrylamide,N,N-dimethylacrylamide and 10-undecenoic acid (98%) were purchased fromAldrich, and were purified by crystallization (n-hexane) andreduced-pressure distillation, respectively. The chain transfer agent(CTA), 2-aminoethanethiol hydrochloride (AET.HCl), was purchased fromSigma, Aldrich. Trinitrobenzene sulfonate (TNBS) 1 M aqueous solutionwas purchased from Fluka. Doxorubicin hydrochloride was kindly providedby Sun Pharmaceuticals, India. 3-[4,5-Dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT, Duchefa) was used in a 5 mg/mL PBS (pH 7.4)solution for cell quantification. The solution was filtered with a 0.22μm filter to remove blue formazan crystals.

ii) Synthesis

P(NIPAAm-co-DM m-co-UA) polymers with various compositions weresynthesized by the radical copolymerization using the redox coupleammonium persulfate (APS) and 2-aminoethanethiol hydrochloride (AET.HCl)(FIG. 1) [Bokias et al. Macromol. Chem. Phys. 199 (1998) 1387-1392]. Thepolymerization procedure is briefly explained as follows.N-isopropylacrylamide (3.965 g, 34.99 mmol) and N,N-dimethylacrylamide(1.48 g, 14.99 mmol) were dissolved in 10 mL of ultra pure water.Undecenoic acid (0.921 g, 5.0 mmol) was converted into sodium salt byreacting with 5 mL of 4% sodium hydroxide solution, and the clearsolution of sodium salt was added to the N-isopropylacrylamide andN,N-dimethylacrylamide solution. The mixture was purged with purifiednitrogen gas for 15 minutes. APS (0.254 g, 4.0 mol % of the monomerfeed) and AET.HCl (0.244 g, 2.16 mmol, 4.0 mol % of the monomer feed)were dissolved in 5.0 mL of ultra pure water. The solution was added tothe monomer solution slowly with continuous stirring. The reaction wascarried out under nitrogen at 27° C. for 48 hours. Upon completion, thecrude product was precipitated by the addition of excess sodium chlorideand dried under vacuum. The crude product was dissolved in ethanol, andwas dialyzed against ultra pure water followed by ethanol using amembrane with a molecular weight cut-off of 2000 (Spectra/Por). Thepurified product was collected after evaporating ethanol.

The chemical structure of the polymers was characterized by ¹H NMR(Bruker AVANCE 400) and Fourier transform infrared (Perkin ElmerSpectrum 2000, KBr) spectroscopic methods. The molecular weights ofpolymers were determined by gel permeation chromatography (GPC, Waters,polystyrene standards) in THF (elution rate: 1 ml/min) at 25° C.Differential scanning calorimetry (DSC) experiments were performed usinga TA 2920 Modulated DSC instrument (CT, USA) with a ramp speed of 3°C./min. Thermogravimetric analyses were performed using TGA 7 (PerkinElmer, USA).

iii) Acid-Base Titration and Amine Group Determination

Acid-base titration was performed to estimate carboxylic acid groups andpKa of the polymer. Briefly, 100 mg of polymer was dissolved in 10 mL ofultra pure water and titrated with 0.01N NaOH using phenolphthalein asan indicator. The apparent partition coefficient pKa of the polymer wasalso determined by this titration method with continuously measuring pHduring the addition of base. From the graph of pH versus the volume ofbase, pKa was calculated as the pH at half the volume of the base at theequivalence point. The free amine group in the polymer was estimated byspectroscopic determination. A known amount of polymer was dissolved in2.0 mL of sodium hydrogen carbonate aqueous solution (2.0 w/v %)containing 0.01M TNBS. The solution was kept for 2 hours at 40° C.,which was then cooled and diluted to a specific volume. The amount ofamine functional groups derivatized with TNBS in the sample wasdetermined by using a UV-VIS spectrophotometer (UV-2501PC, Shimadzu) at345 nm taking L-alanine as standard.

iv) Transmittance Measurements

The LCSTs of polymers in buffer solutions of different pH values weredetermined by monitoring the optical transmittance change as a functionof temperature. Sample solutions (0.5 wt %) were prepared in bufferssuch as neutralized phthalate buffer (pH 5.0), PBS (pH 6.0, 6.6 and7.4), as well as in alkaline borate buffers with pH 9.0 and 10.0. Allthe buffers were prepared with an ionic strength of 154 mM. Opticaltransmittance of the polymer solutions was measured at 500 nm with theUV-VIS spectrometer with the sample cell thermostated using atemperature-controller (TCC-240A, Shimadzu). The heating rate was set at0.1° C./min. The LCST values of polymer solutions were determined at thetemperatures showing an optical transmission of 50%. The effect ofproteins on the LCST was also investigated in the presence of 10 (w/v) %bovine serum albumin (BSA, as a model protein).

v) Fluorescence Measurements

The CMC values of the polymer in PBS (pH 7.4) were determined byfluorescence spectroscopy using pyrene as a probe. Aliquots of pyrenesolutions (1.54×10⁻⁵ M in acetone, 400 μl) were added to 10 mLvolumetric flasks, and the acetone was allowed to evaporate. Polymersolutions at concentrations ranging from 1.0×10⁻⁵ to 1.0 g/L wereprepared in PBS. 10 mL of the aqueous polymer solutions were then addedto the volumetric flasks containing the pyrene residue. It should benoted that all the sample solutions contained excess pyrene content atthe same concentration of 6.16×10⁻⁷ M. The solutions were allowed toequilibrate for 24 hours at room temperature (20° C.). Fluorescencespectra of the polymer solutions were then recorded on a LS50Bluminescence spectrometer (Perkin Elmer, USA) at room temperature. Theemission spectra were recorded from 350 to 500 nm with an excitationwavelength of 340 nm. Both excitation and emission bandwidths were setat 5 nm. From the pyrene emission spectra, the intensity (peak height)ratio (I₃/I₁) of the third band (391 nm, I₃) to the first band (371 nm,I₁) was analyzed as a function of polymer concentration. The CMC valuewas taken from the intersection of the tangent to the curve at theinflection with the horizontal tangent through the points at lowconcentrations.

vi) Preparation of Blank and Drug-Loaded Core-Shell Nanoparticles

The blank core-shell nanoparticles were prepared to investigate theeffect of pH and temperature on the size of the nanoparticles. Thepolymer was dissolved in dimethylacetamide (DMAc) at a concentration of0.5 (w/v) %, which was then dialyzed against 0.02 wt % HCl, and 0.02 wt% NaOH for 24 hours using a membrane with a molecular weight cut-off of2000 (Spectra/Por) at room temperature, respectively. The resultantnanoparticle solutions were freeze-dried after being filtered with a0.45 μm syringe filter and stored at 4.0° C. prior to further analyses.DOX was loaded in the core-shell nanoparticles using a similar protocolas reported by F. Kohori et al. [supra]. Briefly, 7.5 mg of DOX wasneutralized with two moles excess triethylamine in 3 mL of DMAc and thesolution was stirred to dissolve the drug. 15 mg of polymer was thendissolved in the solution. The mixture was dialyzed against 500 mL ofde-ionized water for 48 hours. The DOX-loaded nanoparticles werefiltered and freeze-dried. To determine DOX loading level, a knownamount of DOX-loaded nanoparticles was dissolved in 1 mL of methanol andthen diluted with PBS. The DOX concentration was estimated by using theUV-VIS spectrophotometer at 485 nm. The drug loading was calculatedbased on the standard curve obtained from DOX in PBS (pH 7.4).

vii) Dynamic Light Scattering (DLS) Analyses

The size of the core-shell nanoparticles fabricated at different pH wasanalyzed using ZetaPals (Brookhaven instruments corporations, CA, USA)equipped with a He—Ne laser beam (670 nm). Each measurement was repeatedfive times, and was found to be in a good agreement. An average valuewas obtained from the five measurements. The size of the nanoparticleswas also measured at various temperatures to study the phasereversibility of the nanoparticles. The stability of the re-dispersedfreeze-dried nanoparticles was monitored by measuring their size in PBS(pH 7.4) containing 10 (w/v) % bovine serum albumin (BSA).

viii) Transmission Electron Microscopy (TEM) Examinations

The morphology of the core-shell nanoparticles was analyzed by TEM. Adrop of the freshly prepared nanoparticle solution containing 0.01 (w/v)% phosphotungstic acid was placed on a copper grid coated with a polymerfilm, and was air-dried at room temperature. The TEM observations werecarried out on a JEM-2010 microscope with an electron kinetic energy of200 k eV.

ix) Cytotoxicity Study

Polymer solutions were prepared at stock concentrations. These solutionswere sterilized with 0.22 μm syringe filters and diluted with PBS (pH7.4) and growth media to give the polymer at final concentrations of 10,100, 300 and 400 μg mL⁻¹. Poly(L-lysine) and PEG (Mw 8,000) at aconcentration of 33.3 μg mL⁻¹ were used as the positive and negativecontrols, respectively. PBS (pH 7.4) was used for the blank sampleinstead.

The L929 mouse fibroblast cells were cultured in supplemented Dulbecco'sModified Eagle's Medium (DMEM, 10% fetal bovine serum, 1% L-glutamate,1% penicillin-streptomycin) (GibcoBRL) and incubated at 37° C., 5% CO₂.The cells were seeded onto 96-well plates at 10,000 cells per well. Theplates were then returned to the incubator and the cells were allowed togrow to confluence. On the morning of the initiation of the tests, themedia in the wells were replaced with 150 μl of the pre-prepared growthmedium-sample mixture. The plates were then returned to the incubatorand maintained in 5% CO₂, at 37° C., for 24, 48 and 72 hours. Themixture in each well was replaced with fresh aliquots every morning forthe exposure period. Each sample was tested in eight replicates perplate. Three plates were used for each period of exposure, making atotal of 24 replicates per sample.

Fresh growth media and 20 μL aliquots of MTT solution were used toreplace the mixture in each well after the designated period ofexposure. The plates were then returned to the incubator and maintainedin 5% CO₂, at 37° C., for a further 3 hours. The growth medium andexcess MTT in each well were then removed. 150 μl of DMSO was then addedto each well to dissolve the internalised purple formazan crystals. Analiquot of 100 μL was taken from each well and transferred to a fresh96-well plate. The plates were then assayed at 550 nm and 690 nm. Theabsorbance readings of the formazan crystals were taken to be that at550 nm subtracted by that at 690 nm. The results were expressed as apercentage of the absorbance of the blank, which comprised PBS of acomparative volume, added to the growth medium.

x) In Vitro Drug Release Studies

DOX release from the nanoparticles was studied at pH 6.0, 6.6 and 7.4. Acertain amount of DOX-loaded freeze-dried nanoparticles was dispersed in200 μL of the respective buffer solution and allowed to stabilize for 30minutes before being placed in a dialysis membrane with a molecularweight cut-off of 2000 (Spectra/Por). The dialysis bag was then immersedin 25 mL of PBS with pH 6.0, 6.6 or 7.4 at 37° C. The samples were drawnat specific time intervals and the drug concentration was analyzed usingthe UV-VIS spectrophotometer as stated in paragraph A(vi) of the presentexample.

B) Results and Discussion

i) Polymer Synthesis and Characterization.

A summary on the synthesis and characterization of the copolymers isgiven in Table 1. In these reactions, the feed molar ratios of NIPAAm toDMAAm varied but the content of 10-undecenoic acid was fixed. The CTAwas used at 0.2 and 0.4 mol % of the monomer feed. The polymerizationwas initiated by the thiol radicals, created from the reaction ofAET.HCl with persulphate ions, according to the following equation:2RSH+S₂O₈ ⁻²→2RS+2HSO⁻ ₄where R represents the aminoethyl group. Furthermore the thiol groupsare known to be effective chain transfer agents [Greeg et al., J. Am.Chem. Soc. 70 (1948) 3740-3743]. Thus in this case, the length of theproduced chain is controlled by the molar ratio of the AET.HCl to themonomer feed and the efficiency to initiate polymerization and to dochain transfer reaction. This initiation mechanism by the thiol radicalin redox systems is well established [Khune et al., Polym. Prpr. 22(1981) 76-77]. In addition, for our polymers prepared with thisinitiator couple, the average number of amine functional groups in eachpolymer molecule was estimated to be 1.3 to 1.7. The results wereslightly over estimated probably due to the fact that the averagemolecular weight of polymer was taken for the calculation. A furthermore decrease in the pH of the reaction medium was observed, whichindicates the production of acidic HSO⁻ ₄. The molecular weightsdetermined by GPC indicate that an increased CTA content yielded adecrease in molecular weight, which was in agreement with the resultsreported by G. Bokias et al. [supra].

The ¹H NMR spectra of all the three polymers shared a similar pattern. Atypical ¹H NMR spectrum of Polymer III (NIPAAm:DMAAm:UA=3.50:1.50:0.50)in CDCl₃ is shown in FIG. 2. The success of the copolymerization ofNIPAAm, DMAAm and 10-undecenoic acid in the presence of the chaintransfer agent was evidenced by the absence of vinylic proton signals atδ 5.4-6.6. The broad peaks at δ 1.5-1.8 (Signal a+a′) and at δ 2.1-2.4(Signal b+b′) were attributed to the protons of —CH₂— and —CH— groups inthe NIPAAm and DMAAm moieties, respectively. Other proton signals fromiso-propyl groups (—CHMe₂ at δ 4.0 and —CHMe₂ at δ 1.15, Signals d ande, respectively) and —NMe₂ groups at δ 2.9 (Signal f) were alsoobserved, and their chemical shifts were similar to those of themonomers. From the integration ratio of Signal e to Signal f, the m/nratio was estimated, which was approximately equal to the feed ratio ofthe two monomers. This means that the two monomers had similarreactivity in the polymerization reactions. The FT-IR spectrum ofPolymer III is shown in FIG. 3. It exhibited strong absorptions at about1647 cm⁻¹ (ν_(C═O)) and 1548 cm⁻¹ (ν_(C—N)) from NIPAAm and DMAAmsegments. The absorption of ν_(C═O) in the 10-undecenoic acid segmentsappeared at about 1713 cm⁻¹. The content of UA was estimated as 44.2mg/g Polymer II by the acid-base titration analyses (Table 1). The pKaof Polymer II was about 6.8. The polymers exhibited good solubility inboth water and common organic solvents (CHCl₃, CH₂Cl₂, acetone and THFetc.).

ii) LCST of Polymers and the Effects of pH and Proteins

PNIPAAm exhibits a well-defined LCST of 32° C. in water. The LCST can bemodulated via introducing hydrophobic or hydrophilic monomers. Thepolymers synthesized in this study contains poly(10-undecenoic acid) asthe hydrophobic segment. Thus, environmental pH could influence thehydrophobicity of the 10-undecenoic acid segment through the carboxylicacid groups, which could finally affect the LCST of the polymers. FIGS.4 to 6 show the optical transmittance changes of the polymers at aconcentration of 0.5 wt % in buffer solutions of various pH values as afunction of temperature. From the DLS analyses, the polymers in thebuffer solutions self-assembled into core-shell nanoparticles at theconcentration of 0.5 wt %. The LCST of the core-shell nanoparticlesself-assembled from Polymer I with the NIPAAm/DMAAm/UA ratio of4.00:1.00:0.5 at pH 6.0, 6.6 and 7.4 was 32.5, 33.0 and 33.2° C.,respectively (FIG. 4). However, at pH 5.0, the LCST was drasticallyreduced to 27.8° C. In the case of polymer II with an increased lengthof the hydrophilic DMAAm segment (NIPAAm:DMAAm:UA=3.75:1.25:0.5), theLCST of the core-shell nanoparticles at all the pH values was increased(FIG. 5) when compared to polymer I. The pH value had a significanteffect on the LCST of Polymer II nanoparticles. For instance, at pH 9.0and 7.4, the LCST was found to be 40.5 and 38.5° C., respectively, whichwere well above the normal body temperature. However, at pH 6.6 and 5.0,the LCST was reduced to 35.5 and 35.2° C., respectively, which were muchlower than the normal body temperature. If the nanoparticles have awell-separated core-shell structure or the core is rigid enough, theLCST of the nanoparticles should not be affected by the environmental pHsince the pH-sensitive moieties were in the hydrophobic segments. Thecore-shell nanoparticles made from these polymers might be looselypacked. Thus, the core of the nanoparticles might be well accessible tothe external environment. With the increase of pH of the externalenvironment, the carboxylic acid groups in the 10-undecenoic acidsegment was more de-protonated and thus reduced the hydrophobicity ofthe 10-undecenoic acid segment. This might lead to the increase in theLCST of polymers and thus lead to an increased LCST of thenanoparticles. In spite of having a similar content of carboxylic acidgroups in Polymer I and Polymer II, the effect of de-protonation ofcarboxylic acid groups on the pH sensitivity of Polymer II was moresignificant than Polymer I. It may be because Polymer I had a highermolecular weight. The entropy of mixing decreases with an increasedmolecular weight as the thermodynamic phase separation across the LCSTis caused by low entropy of mixing [Stile et al. Biomacromolecules 3(2002) 591-600; Lessard et al., Can. J. Chem. 79 (2001) 1870-1874]. Thisindicates that the molecular weight is an important factor to influencethe pH-sensitivity of polymers.

A further increase in the length of the hydrophilic segment led togreater LCST as shown in FIG. 6. Among the polymers, Polymer III withthe NIPAAM/DMAAm/UA ratio of 3.5:1.75:0.5 provided core-shellnanoparticles of the highest LCST under all the pH conditions, which washigher than the normal body temperature. For instance, the LCST ofPolymer III nanoparticles at pH 11.0, 7.4, 6.6, 6.0 and 5.5 was 43.0,43.0, 41.0, 40.7 and 39.0° C., respectively. The LCST of Polymer IIInanoparticles was also dependent upon pH. However, itstemperature-sensitivity was low. This might be due to the dilutioneffect of DMAAm, that is, the PNIPAAm segments in the copolymer werewell separated and diluted by DMAAm segments at the high molar ratio,which might reduce intramolecular hydrogen bonding between neighboringamide groups of NIPAAm. As a result, the temperature response of thecopolymer was slow [Liu et al., J. App. Poly. Sci. 90 (2003) 3563-3568;Katsumoto et al., J. Phys. Chem. A. 106 (2002) 3429-3435].

The effect of proteins on the LCST was investigated using Polymer II. Asshown in FIG. 7, the presence of 10 wt % BSA did not alter the LCST ofthe core-shell nanoparticles.

These results show that the polymer can be designed with different LCSTvalues above and bellow the normal body temperature in varying pHenvironments. The core-shell nanoparticles self-assembled from all thethree polymers indeed showed pH-dependent LCST, which may bepredominately triggered by the protonation or de-protonation ofcarboxylic acid groups in the hydrophobic segments of polymers. The LCSTof the nanoparticles was very much influenced by the molar ratio ofNIPAAm to DMAAm. In particular, the LCST of Polymer II nanoparticles washigher than the normal body temperature in the physiological environment(pH 7.4) but lower than the normal body temperature in slightly acidicenvironments. This means that the nanoparticles were soluble and stablein the physiological environment but destabilized/aggregated in acidicenvironments. This unique property may be utilized to target drugs totumor tissues or cell interiors where the environment ischaracteristically acidic.

iii) CMC of Polymer II

The CMC is an important parameter to characterize the stability ofcore-shell nanoparticles. Above the CMC, amphiphilic polymer moleculescan self-assemble into core-shell structured nanoparticles. Thehydrophobic microenvironments of Polymer II nanoparticles in water wereinvestigated by fluorescence spectroscopy using pyrene as a probe. Theratio of I₃ to I₁ was monitored as a function of polymer concentration.FIG. 8 shows plot of I₃/I₁ for Polymer II. A higher ratio is obtainedwhen pyrene is located in a more hydrophobic environment [Dong et al.Can. J. Chem. 62 (1984) 2560-2565]. This property of pyrene can beutilized to study core-shell nanoparticle formation and deformation. TheCMC value was determined to be approximately 10.0 mg/L. It is noticedthat the change in I₃/I₁ after the formation of core-shell nanoparticleswas small. This is probably because the core was loosely packed due tothe presence of carboxylic groups and/or insufficient hydrophobicity ofUA segments.

iv) Size Change of Polymer II Nanoparticles Induced by pH andTemperature Change

The size of Polymer II nanoparticles was found to be pH-dependent. In0.02 wt % HCl solution, the mean diameter of Polymer II nanoparticleswas about 319 nm and in 0.02 wt % NaOH solution, the size of thenanoparticles decreased to about 240 nm. A significantly larger size ofnanoparticles formed in the acidic solution indicates that nanoparticlesin the acidic solution contained a higher degree of aggregation due togreater hydrophobicity of UA at low pH. On the other hand, the repulsionof de-protonated carboxylic acid groups at high pH led to a lower degreeof aggregation, resulting in smaller size. The average size of thenanoparticles loaded with DOX was around 160-200 nm with a narrow sizedistribution as shown in FIG. 9. From the TEM picture (FIG. 10), thesize of the nanoparticles was about 50-60 nm in the solid state, whichmight be due to the collapse of the free hydrophilic segments of thepolymer as well as dehydration of the polymer chain. Meanwhile, it wasobserved that the nanoparticles were stable at pH 7.4 at 37° C. (belowthe LCST) and the size was around 265 nm. Heating the solution to 40° C.(above the LCST), the size increased to about 988 nm because ofaggregation. The aggregates re-dispersed and the size reduced to theoriginal level upon cooling. A similar phenomenon was observed for thenanoparticles at pH 6.6. These results further support the fact that thecore-shell nanoparticles were both pH and temperature sensitive. The pHand temperature response was reversible.

The stability of the drug-loaded core-shell nanoparticles wasinvestigated in PBS (pH 7.4) containing 10 (w/v) % BSA. There was aslight size increase (from 104 to 164 nm) after challenged with BSA for7 hours. This initial size increase might be due to the hydration offreeze-dried nanoparticles. After hydration, the size returned back tothe original level and kept unchanged for next three hours. Thisindicates that the nanoparticles were stable in the presence of BSA.

v) Cytotoxicity Study of Polymer II

The L929 cells were exposed to the polymer at concentrations from 10 to400 mg/L (ppm). From FIG. 11, there did not appear to be any significantcytotoxicity of the Polymer II samples as compared to the negativecontrol. By 72 hours, all the samples of Polymer II appeared to be lesscytotoxic than the positive control.

vi) In Vitro Release

Under the fabrication conditions employed in this study, the actualloading level of DOX was about 2.7% in weight. In vitro drug releasestudy was performed in a slightly acidic environment (pH 6.0 and 6.6) tosimulate the pH of the tumor and in the physiological environment (PBS,pH 7.4). The release profiles of DOX are shown in FIG. 12. The drugrelease from the nanoparticles in pH 7.4 at 37° C. was considerably slowwith an initial burst of about 18%. This initial burst might be due todrug molecules present in the shell of the nanoparticles. However, thedrug release was much faster at pH 6.0 and 6.6 at 37° C. About 70% ofthe drug was released within 48 hours of study. In addition, it wasobserved that the drug-loaded nanoparticles were well dispersed in thebuffer at pH 7.4 but aggregated and settled at the bottom of thedialysis bag at pH 6.0 and 6.6. These results show that thenanoparticles were indeed pH sensitive and slight change in pH from 7.4to 6.6 or 6.0 led to the deformation and precipitation of thedrug-loaded core-shell nanoparticles, thereby releasing the encloseddrug content. In addition, the release of DOX from the dialysis bag wasstudied at pH 7.4 and 6.0. There was no significant effect of pHobserved, which further confirms that the pH-dependent release of DOXfrom the nanoparticles is mainly due to the pH responsiveness of thenanoparticles rather than drug solubility.

C) Conclusions

Amphiphilic tercopolymerspoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-10-undecenoicacid) with various compositions was synthesized by free-radical solutionpolymerization with free amine end group. The core-shell nanoparticlesself-assembled from the polymer with the NIPAAm/DMAAm/UA ratio of3.75:1.25:0.5 were of LCST well above the normal body temperature at pH7.4 and much lower than the normal body temperature in slightly acidicenvironments. The polymer did not show significant cytotoxicity for aperiod of up to 72 hours. The DOX-loaded nanoparticles were stable at pH7.4 at 37° C. and the size was about 160-200 nm. However, at pH 6.0 and6.6, the structure of the nanoparticles was deformed, thereby releasingthe enclosed drug molecules. These properties might help in selectiveaccumulation of the nanoparticles and selective release of the drug inacidic tumor tissues. One more advantage of the polymer synthesized isthat the polymer is with a free amine functional group, which mightallow further modification of the polymer by attaching biologicalsignals for active targeting.

Example 2 Synthesis ofpoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-10-undecenoicacid) [P(NIPAAm-co-DMAAm-co-UA)] Having a Folate Targeting Group A)Experimental Section

i) Materials

Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-10-undecenoicacid) [P(NIPAAm-co-DMAAm-co-UA), Polymer II] was synthesized by freeradical polymerization as explained in Example 1. Folic acid dihydrate,N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), dimethylsulfoxide (DMSO) were purchased from Sigma, Aldrich. Paclitaxel waspurchased from Merck.

ii) Conjugation of Folic Acid to P(NIPAAm-co-DMAAm-co-UA) andConjugation of Folic Acid to P(NIPAAm-co-DMAAm-co-UA) Grafted withCholesterol

NHS ester of folic acid (NHS-folate) was prepared by the followingmethod: folic acid (5 gm dissolved in 100 mL of DMSO plus 2.5 mL oftriethylamine) was reacted with N-hydroxysuccinimide (2.6 g) in thepresence of DCC (4.7 g) overnight at room temperature. The by-product,dicyclohexylurea was removed by filtration (as shown in FIG. 13, Scheme2). To conjugate the folic acid to Polymer II (Poly-FA), the activatedNHS-folate in DMSO was added to Polymer II in PBS buffer (pH 7.4) withconstantly stirring for 5 hrs at room temperature (FIG. 14, Scheme 3).The folic acid-conjugated polymer was purified by dialysis in thepresence of PBS buffer (pH 7.4) for 24 hrs and followed by ultra purewater for 24 hrs using dialysis membrane of molecular weight cut-off of2000 (Spectra/Por). The polymer was freeze-dried and stored in anairtight container for further use. Folic acid was also conjugated toPolymer II grafted with cholesterol (Poly-CH-FA). Polymer II graftedwith cholesterol (Poly-CH) was synthesized by reacting Polymer IIactivated with NHS (The procedure was similar to the activation of folicacid by using 1:2:2 molar ratio of polymer II, NHS and DCC respectively)with an equal molar concentration of cholesterol in the hydroalcoholicsolution for 48 hrs at room temperature (FIG. 15, Scheme 4).

Chemical structure of the polymers was characterized by ¹H NMR (BrukerAVANCE 400) and Fourier transform infrared (Perkin Elmer Spectrum 2000,KBr) spectroscopic methods. Differential scanning calorimetry (DSC)experiments were performed using a TA 2920 Modulated DSC instrument (CT,USA) with a ramp speed of 3° C./min.

iii) Preparation of Drug-Loaded Polymer-CH-FA Core-Shell Nanoparticles

Doxorubicin was loaded in the core-shell nanoparticles as follows: 7.5mg or 5.0 mg of DOX was dissolved in 3 mL of DMAc or DMF with stirring.15 mg of polymer was then dissolved in the solution. The mixture wasdialyzed against 500 mL of de-ionized water for 48 hours. To determineDOX loading level, a known amount of DOX-loaded nanoparticles wasdissolved in 1 mL of methanol and then diluted with PBS. The DOXconcentration was estimated by using the UV-VIS. Initial study was alsoperformed by loading the core-shell nanoparticles with paclitaxel, awater insoluble anti-cancer drug. Briefly, 15 mg of the polymer and 2.5mg of paclitaxol was dissolved in 3 mL of DMF, the polymer drug solutionwas dialyzed in the presence of ultra pure water for 24 hrs. Thedrug-loaded nanoparticles were filtered through a disc filter of 0.45 μmpore size and freeze-dried. To determine loading level of paclitaxol,paclitaxol was extracted from the polymeric nanoparticles by dissolvingthe nanoparticles in 1 mL of chloroform, and the polymer wasprecipitated by adding 2 mL of diethyl ether. After centrifuge, thesupernatant was collected, dried and analyzed by HPLC (Waters, model2690, C8 15×4.6 cm column). The mobile phase consisted of 20 mM ammoniumacetate, acetonitrile and methanol in the volume ratio of 35:45:20. Thestandard paclitaxol solutions were prepared in methanol withconcentrations ranging from 5 to 100 ppm.

B) Results and Discussion

Folic acid was conjugated successfully to Polymer II and polymer IIgrafted with cholesterol. This is confirmed by NMR studies (FIGS. 16 and17). The success of the conjugation of folic acid was evidenced by thepresence of proton signals at δ 6.6-6.8 and δ 7.5-7.7 from the aromaticprotons 2, 6 and 3, 5 in the folic acid molecule (FIG. 14, Scheme 3).The conjugation of the cholesterol on to Polymer II was also evidencedby the proton signals (δ 0.6-1.1) from the five CH₃ groups ofcholesterol.

From Example 1, the LCST of Polymer II was 38.5° C. at pH 7.4, whichdecreased to 35.5° C. at pH 6.6. This is due to the protonation anddeprotonation of the polymer with the change in pH, which changes thehydrophobicity of the polymer. There was no significant difference inthe LCST at pH 7.4 between Poly-FA and Polymer II. However, there was anincrease in the LCST at pH 6.6, and the temperature sensitivity waslower (FIG. 18). This might be due to the fact that folic acid increasedthe hydrophilicity of the polymer at this pH. However, the solubility ofthe polymer in pH 5.0 was low and the solution was turbid. The LCST ofthe polymer decreased to 36° C. and the temperature sensitivity washigher (data not shown). This might be due to the fact that folic acidhas a pKa around pH 5.4, its carboxylic functional groups wereprotonated at pH 5.0, increasing the hydrophobicity of the polymer. Thisproperty may help in intracellular delivery of drugs due to the LCSTlower than normal body temperature at pH 5.0 (in endosomes), which mayhelp break down the endosome membrane. When cholesterol was conjugatedto Polymer II, the LCST of the polymer was 35.7° C. at pH 7.4 as shownin FIG. 19, which was lower than Polymer II. This is because graftingcholesterol increased the hydrophobicity of the polymer, decreasing theLCST. On the other hand, the LCST of Poly-CH-FA was 39.0 and 34.5° C. atpH 7.4 and pH 6.6 respectively (FIG. 20). This might be due to the factthat the increase in the hydrophobicity of the polymer caused by theintroduction of cholesterol was equally compensated by the folic acidmolecule.

ATCC L929 cells were exposed to the polymer at concentrations from 10 to100 mg/L (ppm). From FIG. 21, there did not appear to be any significantcytotoxicity of the Poly-CH-FA samples as compared to the negativecontrol [poly(ethylene glycol)]. However, all the samples of Poly-CH-FAwere less cytotoxic than the positive control [poly(L-lysine)].

A typical dialysis method was used to prepare Poly-CH-FA empty anddrug-loaded core-shell nanoparticles. Doxorubicine hydrochloride andpaclitaxol were selected as water-soluble and water-insoluble drugs. Theeffect of the solvent on the drug loading of doxorubicine was studied.With the use of DMAc, there was an encapsulation efficiency of 12.9%with drug loading of 4.31% in weight. The average diameter of particleswas about 265 nm. However, in the case of the dialysis method where DMFwas used as the solvent, the drug loading was decreased to 0.6% with anencapsulation efficiency of 2.4% and an average particle size of 100 to160 nm. The decreased drug loading in the later formulation might be dueto the fact that the solubility of the DMF was higher (12.1 cal/cm³)compared to DMAc (10.8 cal/cm³), which might help in the early escape ofthe drug before getting encapsulated. The decreased particle size mightbe attributed to decreased drug loading.

Paclitaxol was loaded into the core-shell nanoparticles with an averageparticle size of 96 nm, an encapsulation efficiency of 13.0% and a drugloading of 1.9%. Paclitaxol is a crystal drug with a melting point of220° C. (FIG. 22). The melting point of paclitaxol disappeared afterencapsulated in the nanoparticles, indicating that the drug wasmolecularly distributed.

C) Conclusion

Core-shell nanoparticles with an active targeting signal (folic acid) totumor cells was synthesised. The nanoparticles retained the pH sensitiveproperty, and possessed low cytotoxicity. Two anticancer drugs wereloaded into the core-shell nanoparticles. The particle size and loadinglevel of drugs can be manipulated by varying fabrication conditions.

Example 3 Synthesis of Doxorubicin Conjugatedpoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-10-undecenoicacid) [P(NIPAAm-co-DMAAm-co-UA)]

i) Conjugation of Doxorubicin to Copolymer

Polymer II can also be conjugated to drugs which are having reactivefunctional groups. Doxorubicin (FIG. 23) was conjugated throughcarbodiimide chemistry wherein the amine functional group of doxorubicinwas conjugated to carboxyl functional groups of polymer II, as presentedin FIG. 24. Briefly, polymer II conjugated with doxorubicin (Poly-DOX)was synthesized by reacting the polymer II activated with NHS (procedurewas similar to activation of folic acid described in Example 2) withdoxorubicin (its concentration was two times as high as that of polymerII.) in phosphate buffer (pH 7.4) for 48 hrs at room temperature. Theblood red colored product was obtained after dialyzing in the presenceof ultra pure water for 48 hours using a dialysis membrane with a 2000molecular weight cut-off, followed by freeze-drying. It is confirmedfrom the gel permeation chromatography that there was an increase in themolecular weight of the polymer II from Mw: 9,051, Mn: 6,781 to Mw:11,129, Mn: 9,118, and also a decrease in the retention time as shown inFIG. 25. Moreover, the differential scanning calorimetry of theconjugate shows that there was no appearance of transition for meltingpoint of doxorubicin at 202° C. (as shown in FIG. 26), which indicatesthat drug was part of the polymer chain.

ii) Fabrication of Micelles from the Doxorubicin Conjugated Polymer II

After conjugation of the drug on to the polymer chain, it was observedthat the polymer was relatively insoluble in water. It was attempted toprepare core-shell nanoparticles (micelles) using this polymer by bothdialysis as well as solvent evaporation methods. It was found thatdialysis was not a suitable method as the polymer is more hydrophobic,leading to the precipitation of the polymer or formation of biggerparticles in the range of 800-1000 nm. However, by using the solventevaporation method, it was able to produce micelles with a mean diameterof 280 nm. Procedure for solvent evaporation was as follows: 15 mg ofthe conjugated polymer was dissolved in 4 mL of dimethylacetamide and 1mL of dichloromethane, and the polymer solution was emulsified into 20mL of ultra pure water and sonicated for 5 mins. The solvent wasevaporated and the solution was centrifuged and measured for particlesize. Conjugation of the drug to the micelle did alter the pH triggeredtemperature sensitivity of the polymer. It was observed that there was adecrease in the particle size above 38° C. (above the LCST), which mightbe due to the collapse of the temperature sensitive segment in pH 7.4.This phenomenon was found to be reversible and reproducible (FIG. 27).

Example 4 Synthesis of Block Copolymerpoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(10-undecenoicacid) [P(NIPAAm-co-DMAAm)-b-PUA]

The synthesis of a block copolymer of polymer II was carried out bysynthesizing the temperature sensitive/hydrophilic segment and pHsensitive fatty acid segment separately, and they were then conjugatedto produce the block co-polymer as shown in FIGS. 28 and 29 (Scheme 5and 6). Briefly, the temperature sensitive segment was synthesized byreacting purified N-isopropylacrylamide) and N,N-dimethylacrylamide in amonomer ratio of 3.75:1.25, with 0.4 mol % of the chain transfer agent,2-aminoethanethiol hydrochloride (AET.HCl) in 40 mL of alcohol in thepresence of initiator azobisisobutyronitrile at 70° C. for 24 hrs. Thepolymer was purified by dissolving it in chloroform and precipitating itin diethyl ether. Molecular weight of the polymer was analyzed by GPC,and found to be Mw 11,221, On the other hand, poly(10-undecenoic acid)was synthesized by reacting the sodium salt of monomer (0.097 mol) inthe presence of ammonium persulfate (0.8 g) in water at 70° C. for 24hr. The polymer was precipitated in the presence of cold ethanol.Poly(10-undecenoic acid) was activated by NHS in the presence of DCC,and this product was further conjugated to temperature sensitive blockin water at alkaline pH. Polymer molecular weight was Mw 29,177 (FIG.30). The block co-polymer was analyzed for their pH and temperaturesensitivity by measuring the LCST. As shown in FIG. 31, it is confirmedthat the block co-polymer was indeed pH and temperature sensitive. TheLCST of the block copolymer in PBS (pH 7.4) was 39.5° C., which reducedto 38.5° C. when blocked with poly(10-undecenoic acid). The blockco-polymer exhibited a LCST of 36.7° C. at pH 6.0.

What is claimed is:
 1. An amphiphilic block copolymer comprising atleast three types of monomeric units, said three types of monomericunits comprising: a temperature-sensitive monomeric unit, wherein thetemperature-sensitive monomeric unit imparts temperature-induced phaseseparation character to the amphiphilic block copolymer, a hydrophilicmonomeric unit, wherein the hydrophilic monomeric unit has one or morepolar functionalities, and a hydrophobic monomeric unit comprising atleast one pH-sensitive moiety; wherein said hydrophobic monomeric unitis derived from a copolymerisable unsaturated fatty acid; wherein saidunsaturated fatty acid imparts hydrophobic character to the amphiphilicblock copolymer; wherein the fatty acid comprises 5 to 50 or more mainchain carbon atoms; wherein the fatty acid comprises one carbon-carbondouble bond (monounsaturated); and wherein the amphiphilic blockcopolymer exhibits a lower critical solution temperature (LCST)dependent on environmental pH.
 2. The amphiphilic block copolymer ofclaim 1, wherein the fatty acid is selected from the group consisting of(E)-9-Octadecenoic acid, (Z)-9-Octadecenoic acid, (Z)-11-Octadecenoicacid, (E)-9-Hexadecenoic acid, (Z)-9-Hexadecenoic acid,(Z)-9-Tetradecenoic acid, (Z)-11-Eicosenoic acid, (Z)-13-Docosenoic Acidand (Z)-15-Tetracosaenoic Acid.
 3. The amphiphilic block copolymer ofclaim 1, wherein the fatty acid is an omega-1 fatty acid.
 4. Theamphiphilic block copolymer of claim 3, wherein the fatty acid isselected from the group consisting of 4-pentenoic acid, 7-octenoic acid,10-undecenoic acid, 15-hexadecenoic acid, and 19-eicosenoic acid.
 5. Theamphiphilic block copolymer of claim 1, wherein the temperaturesensitive monomeric unit is derived from the group consisting ofN-acroylpiperadine, N-t-butylacrylamide, N-piperidyl-methacrylamide andN-isopropylacrylamide.
 6. The amphiphilic block copolymer of claim 1,wherein the hydrophilic monomeric unit is derived from the groupconsisting of acrylic acid, acrylamide, acrylate, and substitutedderivatives thereof.
 7. The amphiphilic block copolymer of claim 6,wherein the acrylamide is selected from the group consisting ofacrylamide (AAm), N,N′-dimethylacrylamide (DMAAm), andN-(hydroxymethyl)acrylamide.
 8. The amphiphilic block copolymer of claim1, further comprising a terminal group comprising at least one moietyselected from the group consisting of a terminating moiety, a ligand, adrug, a tag, a radioimmunoconjugate, a chemical moiety and a spacer. 9.The amphiphilic block copolymer of claim 8, wherein the terminatingmoiety comprises a functional group selected from the group consistingof a hydroxyl group, a carboxyl group and an amino group.
 10. Theamphiphilic block copolymer of claim 9, wherein the terminating moietyis introduced by chain transfer agents or group transfer agents.
 11. Theamphiphilic block copolymer of claim 9, wherein the terminating moietyis introduced by living polymerisation methods.
 12. The amphiphilicblock copolymer of claim 9, wherein the terminating moiety is part ofthe monomeric unit of the polymer.
 13. The amphiphilic block copolymerof claim 10, wherein the chain transfer agent is selected from the groupconsisting of chloroform, carbon tetrachloride, alkyl-mercaptans,aminoethanethiol, mercapto-propionic acid, mercapto-succinic acid,thioglycolic acid, mercaptoethanol and secondary alcohols ofmercaptoethanol, alkyl halides, and salts of phosphorus acids with anoxidation number less than
 5. 14. The amphiphilic block copolymer ofclaim 13, wherein the alkyl-mercaptans are selected from the groupconsisting of octanethiol, decanethiol, n-dodecanethiol ort-dodecanethiol.
 15. The amphiphilic block copolymer of claim 8, whereinsaid ligand is attached to the functional group of the terminatingmoiety directly.
 16. The amphiphilic block copolymer of claim 8, whereinsaid ligand is attached to the terminating moiety by a spacer.
 17. Theamphiphilic block copolymer of claim 16, wherein said spacer comprisesmore than 10 main chain atoms.
 18. The amphiphilic block copolymer ofclaim 17, wherein said ligand is selected from the group consisting ofsmall targeting molecules, proteins, peptides and carbon hydrates. 19.The amphiphilic block copolymer of claim 17, wherein the spacercomprises poly(ethylene glycol) and poly(propylene glycol).
 20. Theamphiphilic block copolymer of claim 1, wherein the copolymer is arandom copolymer.